Helmut G. Rennke

Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension.

Two groups of adult male Munich-Wistar rats and a third group of nondiabetic age-matched and weight-matched normal control rats underwent micropuncture study 1 mo, and morphologic studies 14 mo, after induction of streptozotocin diabetes or sham treatment. All animals were fed standard rat chow. Diabetic rats received daily ultralente insulin to maintain stable moderate hyperglycemia (approximately 350 mg/dl). In addition, one group of diabetic rats was treated with the angiotensin I converting enzyme inhibitor, enalapril, 15 mg/liter of drinking water. Average kidney weight, whole kidney and single-nephron glomerular filtration rate, and glomerular plasma flow rate were elevated to similar values in both groups of diabetic rats, relative to normal control rats. Non-enalapril-treated diabetic rats exhibited significant elevations in mean glomerular capillary hydraulic pressure and transcapillary hydraulic pressure gradient, compared with the other groups studied, and only this group eventually developed marked and progressive albuminuria. Likewise, histological examination of the kidneys at 14 mo disclosed a high incidence of glomerular structural abnormalities only in non-enalapril-treated diabetic rats. These findings indicate that prevention of glomerular capillary hypertension in rats with diabetes mellitus effectively protects against the subsequent development of glomerular structural injury and proteinuria. This protection is afforded despite pronounced hyperglycemia and elevated levels of glucosylated hemoglobin, further supporting our view that hemodynamic rather than metabolic factors predominate in the pathogenesis of diabetic glomerulopathy.

Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding α-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant α-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type α-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.

Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat.

Micropuncture and morphologic studies were performed in six groups of male Munich-Wistar rats after removal of the right kidney and segmental infarction of two-thirds of the left kidney. Groups 1 and 4 received no specific therapy. Groups 2 and 5 were treated with the angiotensin I-converting enzyme inhibitor, enalapril, 50 mg/liter, in the drinking water. Groups 3 and 6 were treated with reserpine (5 mg/liter), hydralazine (80 mg/liter), and hydrochlorothiazide (25 mg/liter). All rats were fed standard chow. Groups 1-3 underwent micropuncture study 4 wk after renal ablation. Untreated group 1 rats exhibited systemic hypertension and elevation of the single nephron glomerular filtration rate (SNGFR) due to high average values for the mean glomerular transcapillary hydraulic pressure gradient (delta P) and glomerular plasma flow rate (QA). In group 2 rats, treatment with enalapril prevented systemic hypertension and maintained delta P at near-normal levels without significant reduction in SNGFR and QA. In contrast, triple drug therapy normalized systemic hypertension, but failed to lower delta P in group 3 rats. Groups 4-6 were followed for 12 wk after renal ablation. Untreated group 4 rats demonstrated continuous systemic hypertension, progressive proteinuria, and glomerular structural lesions, including mesangial expansion and frequent areas of segmental sclerosis. In group 5 rats, treatment with enalapril maintained systemic blood pressure at normal levels over the 12-wk period and dramatically limited the development of proteinuria and glomerular lesions. Despite equivalent systemic blood pressure control in group 6 rats, failure of triple drug therapy to control glomerular hypertension was associated with progressive proteinuria and glomerular lesions comparable to those seen in untreated group 4 rats. Thus, unless glomerular capillary hypertension is corrected, control of systemic blood pressure is insufficient to prevent progressive renal injury in rats with reduced renal mass.

Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass.

Micropuncture and morphologic studies were performed in four groups of male Munich-Wistar rats after removal of the right kidney and segmental infarction of two-thirds of the left kidney. Groups 1 and 3 received no specific therapy. Groups 2 and 4 were treated with the angiotensin I converting enzyme inhibitor, enalapril, 50 mg/liter of which was put in their drinking water. All rats were fed standard chow. Groups 1 and 2 underwent micropuncture study 4 wk after renal ablation. Untreated group 1 rats exhibited systemic hypertension and elevation of the single nephron glomerular filtration rate (SNGFR) due to high average values for the mean glomerular transcapillary hydraulic pressure difference and glomerular plasma flow rate. In group 2 rats, treatment with enalapril prevented systemic hypertension and maintained the mean glomerular transcapillary hydraulic pressure gradient at near-normal levels without significantly compromising SNGFR and the glomerular capillary plasma flow rate, as compared with untreated group 1 rats. Groups 3 and 4 were studied 8 wk after renal ablation. Untreated group 3 rats demonstrated persistent systemic hypertension, progressive proteinuria, and glomerular structural lesions, including mesangial expansion and segmental sclerosis. In group 4 rats, treatment with enalapril maintained systemic blood pressure at normal levels over the 8-wk period and significantly limited the development of proteinuria and glomerular lesions. These studies suggest that control of glomerular hypertension effectively limits glomerular injury in rats with renal ablation, and further support the view that glomerular hemodynamic changes mediate progressive renal injury when nephron number is reduced.

Podocyte loss and progressive glomerular injury in type II diabetes.

Kidney biopsies from Pima Indians with type II diabetes were analyzed. Subjects were classified clinically as having early diabetes (n = 10), microalbuminuria (n = 17), normoalbuminuria, despite a duration of diabetes equal to that of the subjects with microalbuminuria (n = 12), or clinical nephropathy (n = 12). Subjects with microalbuminuria exhibited moderate increases in glomerular and mesangial volume when compared with those with early diabetes, but could not be distinguished from subjects who remained normoalbuminuric after an equal duration of diabetes. Subjects with clinical nephropathy exhibited global glomerular sclerosis and more prominent structural abnormalities in nonsclerosed glomeruli. Marked mesangial expansion was accompanied by a further increase in total glomerular volume. Glomerular capillary surface area remained stable, but the glomerular basement membrane thickness was increased and podocyte foot processes were broadened. Broadening of podocyte foot processes was associated with a reduction in the number of podocytes per glomerulus and an increase in the surface area covered by remaining podocytes. These findings suggest that podocyte loss contributes to the progression of diabetic nephropathy.

The case for intrarenal hypertension in the initiation and progression of diabetic and other glomerulopathies

G ross defects in glomerular capillary function manifested as heavy proteinuria and severely reduced glomerular filtration rates occur with long-standing diabetes mellitus. However, substantial changes in glomerular function appear even at an early phase of this metabolic disorder, and studies of these early alterations have begun to yield insights into potential mechanisms of the late, overt injury. In 1959, Stalder and Schmid [l] demonstrated supernormal glomerular filtration rate values in diabetic children and young adults, a finding confirmed by Ditzel and Schwartz [2] and extensively investigated in recent years by Mogensen [3,4]. Mogensen [3] described a 40 percent increment in glomerular filtration rate in 11 patients with newly diagnosed juvenile diabetes when compared to values in 31 normal subjects of similar age. This remarkable hyperfiltration was shown to be related to the patient’s metabolic status, since reduction of blood sugar levels over several days to weeks by standard insulin therapy tended to return glomerular filtration rate to normal or near-normal values. Indeed, recent studies by Christiansen et al. [5] have demonstrated that with reduction in blood glucose levels to normal by continuous insulin infusion, glomerular filtration rate also declines from elevated to near-normal values in a matter of hours. A number of clinical studies have been performed, largely by these and other Danish investigators, in an attempt to dissect the particular component or components of the diabetic state responsible for these early elevations in glomerular filtration rate. One factor that has been incriminated is hyperglycemia per se. Indeed, acute elevations of glomerular filtration rate occur in response to glucose infusion in normal subjects [ 6-81. While the structural hypertrophy observed in kidneys of diabetic humans and animals, best documented by

Hemodynamic basis for glomerular injury in rats with desoxycorticosterone-salt hypertension.

sterby, Gunderson and co-workers [ 9, lo], has also been proposed to contribute to the augmented glomerular filtration rate, this process would be unlikely to explain the relatively rapid changes in glomerular filtration rate that occur with insulin therapy. Augmented levels of glucagon and growth hormone are usual accompaniments of diabetic hyperglycemia, and each of these hormones is capable of inducing a rise in glomerular filtration rate in normal subjects at plasma levels comparable to those found in hyperglycemic diabetic patients [ 1 l-141. However, increments in glomerular filtration rate achieved by these hormones are substantially less than the usual increment observed in the early insulin-dependent diabetic patient. Finally, changes in circulating and tissue levels of classic vasoactive hormones such as angiotensin II, catecholamines and prostaglandins have been demonstrated in a variety of circumstances in diabetes [ 1% 171. Also, changes in responsiveness to angiotensin II and catecholamines have been demonstrated in several extrarenal vascular beds in diabetic patients and animals [ 18,191. Whether changes in the levels of these hormones or the vascular response to them contribute to the hyperfiltration is as yet unknown. In any case, a variety of factors present in the diabetic state may contribute to hyperfiltration, and no single factor appears to account fully for this phenomenon. Although insights into the stimuli responsible for diabetic hyperfiltration have been forthcoming from such

Pathogenesis and Significance of Nonprimary Focal and Segmental Glomerulosclerosis

Micropuncture and/or morphologic studies were performed in seven groups of uninephrectomized (UNX) adult male Munich-Wistar rats. Control groups 1, 3, and 6 received standard (24% protein) chow and tap water. Groups 2, 4, and 5 received weekly injections of desoxycorticosterone pivilate (DOC) and 1% saline for drinking, groups 2 and 4 were fed standard chow, and Group 5 a diet containing 6% protein. Group 7 received DOC, salt, and standard chow for 3 wk followed by withdrawal of DOC and salt for an additional 6 wk. 10-14 d after UNX, groups 1 and 2 exhibited similar single nephron glomerular filtration rates (SNGFR) and initial glomerular plasma flow rates (QA). Group 2 had higher mean arterial pressure (AP) and glomerular capillary hydraulic pressure (PGC) than group 1. 3-4 wk after UNX, group 4 exhibited further elevations in AP and PGC as compared with groups 2 and 3. SNGFR and QA were similar in groups 3 and 4, but these average values were greater than typical for normal rats. Group 4 also demonstrated increased urinary protein excretion. Morphologic evaluation of glomeruli in groups 2 and 4 revealed mesangial expansion and focal intraglomerular hemorrhage whereas glomeruli of groups 1 and 3 were essentially normal. Values for AP and PGC in group 5 were not different than group 3 but significantly lower than group 4. QA and SNGFR were lower in group 5 (low protein) than in groups 3 and 4. Furthermore, proteinuria and glomerular structural lesions were abolished in group 5. Morphologic studies performed in groups 6 and 7 showed that early DOC-SALT lesions progress to focal glomerular sclerosis. These studies suggest that continued elevations in glomerular capillary flows and pressures predispose to glomerular injury in this model of systemic arterial hypertension.

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

Injury of the glomerular microvasculature by nonimmunologic processes is often the underlying mechanism of progressive deterioration of renal function in patients with a variety of renal disorders. The structural hallmark of this injury is focal and segmental glomerulosclerosis, often accompanied by entrapment of hyalin. Although such lesions are quite characteristic for diseases that primarily affect the glomerular podocyte, similar damage occurs in association with functional and structural adaptive changes that develop as a consequence of a significant loss of functioning nephrons or other systemic disorders. Experimental studies have revealed that such functional adaptations include intrarenal vasodilatation that through increases in glomerular capillary pressure and plasma flow leads to a significant compensatory hyperfiltration. This functional state is accompanied by a parallel increase in glomerular volume, attained chiefly by expansion of matrix components and an increase in the number of endothelial and mesangial cells, but not of podocytes. The persistence of the adaptive changes results in endothelial, mesangial, and epithelial cell dysfunction revealed clinically by proteinuria and structurally by the development of microthrombosis, microaneurysms, mesangial expansion, and occlusion of capillaries by hyalin accumulation. Although all these pathologic processes can lead to segmental collapse of the capillary tuft, it is the progressive hyalin deposition in capillaries with defective or detached podocytes that represents the major mechanism in the development of segmental and eventually global glomerulosclerosis. The inability of the highly differentiated podocyte to replicate in response to systemic or locally released trophic factors ultimately results in imperfections of the capillary wall that set the stage for permeability defects amplified and accentuated by greatly augmented hydrodynamic forces. These structural and functional microvascular changes acting in concert not only facilitate the transcapillary convection of macromolecules that results in albuminuria, but can also be anticipated to play a key role in the entrapment and accumulation of larger macromolecules in front of the lamina densa in the form of hyalin material. Continuing damage to the glomerular microvasculature exacerbates the adaptive changes in surviving nephrons, closing a positive-feedback loop that culminates in end-stage renal failure.

Identification and Localization of Polycystin, the PKD1 Gene Product

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henles loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.